Involvement of RhoA/Rho-associated kinase signal transduction pathway in dexamethasone-induced alterations in aqueous outflow

• Published in: Investigative ophthalmology & visual science Vol. 53; no. 11; pp. 7097 - 7108
• Main Authors: Fujimoto, Tomokazu, Inoue, Toshihiro, Kameda, Takanori, Kasaoka, Nanako, Inoue-Mochita, Miyuki, Tsuboi, Naoko, Tanihara, Hidenobu
• Format: Journal Article
• Language: English
• Published: United States 11.10.2012
• Subjects: Actins - metabolism; Amides - pharmacology; Animals; Anterior Chamber - metabolism; Aqueous Humor - metabolism; Cell Survival - drug effects; Cell Survival - physiology; Cells, Cultured; Dexamethasone - pharmacology; Enzyme Inhibitors - pharmacology; Extracellular Matrix Proteins - genetics; Glucocorticoids - pharmacology; Integrins - genetics; Intercellular Junctions - drug effects; Intercellular Junctions - physiology; Myosin Light Chains - metabolism; Organ Culture Techniques; Phosphorylation - drug effects; Phosphorylation - physiology; Pyridines - pharmacology; rho-Associated Kinases - metabolism; rhoA GTP-Binding Protein - metabolism; RNA, Messenger - metabolism; Signal Transduction - drug effects; Signal Transduction - physiology; Swine; Trabecular Meshwork - cytology; Trabecular Meshwork - drug effects; Trabecular Meshwork - physiology
• ISSN: 1552-5783; 1552-5783
• DOI: 10.1167/iovs.12-9989

We investigated the involvement of the RhoA/Rho kinase (ROCK) signal transduction pathway in dexamethasone (DEX)-induced changes in aqueous outflow. Using trabecular meshwork (TM) and Schlemm's canal endothelial (SCE) cells, RhoA activation was evaluated with a pull-down assay and myosin light chain phosphorylation was evaluated by Western blot analysis. Outflow facility was measured in perfused porcine anterior segment organ cultures treated with DEX and/or Y-27632, a selective ROCK inhibitor. The barrier function of the cultured cells on a micropore filter was evaluated by measuring the transendothelial electrical resistance. Collagen, fibronectin, and integrin mRNA expression levels were evaluated by quantitative real-time RT-PCR. Relative RhoA activities increased following stimulation with 100 nM DEX in TM and SCE cells. Perfusion with DEX decreased outflow facility by 31.9 ± 14.3% compared to controls at 24 hours, but not by 50 μM Y-27632 in addition to DEX. The transendothelial electrical resistance of the SCE cell monolayer was increased by 48.6 ± 6.4% and 5.3 ± 5.0% following DEX treatments without and with 10 μM Y-27632, respectively, compared to controls. In TM cells, the mRNA expressions of COL4A1 and fibronectin were increased significantly by DEX treatment, but combined treatment with Y-27632 and DEX significantly inhibited the increase in COL4A1 and fibronectin expression. Activation of the Rho/ROCK pathway in SCE cells contributes to the mechanism of DEX-induced changes in aqueous outflow.